Immune complexes are formed by the binding of antibodies with antigens, sometimes in conjunction with other proteins. The antigens which form immune complexes include components of infectious organisms, other molecules foreign to the host organism, tumor-associated molecules and, in many diseases, normal tissue molecular components. Antibodies produced to an antigen are specific for the particular antigenic substance. Antibodies bind to antigens and essentially neutralize them by, e.g., altering the biologic activity of a toxin, neutralizing the infectivity of microorganisms, or by providing the recognition signal whereby the antibody plus bound antigen are removed from the circulation or tissues. When an antibody binds to an antigen, the antibody-antigen complex is termed an immune complex. ##EQU1##
Immune complexes are removed from the circulation and tissues by a variety of normal mechanisms such as by fixed macrophages found in the liver, spleen and lymph nodes, and by circulating macrophages. The formation of antibodies and immune complexes is part of the natural response of the individual to combat diseases, such as infections and cancer. The binding of antibodies to antigens and the removal of the immune complexes are the mechanisms by which antigens are neutralized, taken out of tissues and blood and degraded.
The continued presence of immune complexes in the circulation and their deposition in tissues, contributes to compromised immune system function and inflammatory pathology. Immune complexes can deposit in tissues such as the lung, kidney, heart and joints causing both transient and permanent damage to those organs. In cancer, it is thought that the immune complexes may block the proper function of immune mechanisms which would otherwise destroy the cancer cells and prevent the growth and spread of the cancer within the body.
There may be one or several reasons why circulating immune complexes are found in high levels in diseases. The antigens and the immune system responses may be near maximal, and the immune complexes formed may simply overwhelm the capacity of the systems for their removal. Alternatively, some mechanism may have compromised the efficiency of the immune complex removal system, or the nature of the antigens and antibodies involved may result in inefficient removal. Immune complexes are found to persist in many individuals with cancer, autoimmune, arthritic, and infectious diseases. Therefore, the binding and removal of immune complexes, by an extracorporeal device, may result in a clinical improvement of individuals with cancer and improve the effectiveness of other treatments for cancer, may result in the prevention of lesions in organs associated with immune complexes that occur in infectious diseases, and may prevent lesions in organ systems in arthritic and autoimmune diseases, as well as result in the clinical improvement of these latter diseases. Indeed, many published studies indicate that the removal of circulating immune complexes, or enhancing their clearance from the circulation, may constitute an effective therapeutic treatment. [See Theofilopoulos, A. M. and Dixon, F. J., Adv. Immunol., 28:90-220 (1979); Theofilopolous, A. N. and Dixon, F. J., Immunodiagnostics of Cancer, p. 896 (M. Decker Inc., New York 1979)].
There can be a wide variety in the number of anti-body molecules which bind to antigen, and antigens are of various sizes and shapes, thus leading to wide variations in the sizes of the immune complexes. Some immune complexes can also affix complement proteins such as C1q and C3 which may result in larger, more heterogeneous structures. Some immune complexes stimulate leukocytes while others stimulate lymphocytes or platelets [See Ritzmann, et al., Clin. Chem., 28:1259-71 (1982); Maire, et al., Clin. Exp. Immunol., 51:215-224 (1983), and Schifferli, et al., New Eng. J. Mad., 315:448-495 (1986)]. Immune complexes may remain in circulation for long periods of time and deposit in various tissues contributing to the inflammatory and erosive manifestations of autoimmune and other diseases [See Emancipator, et al., Lab. Invest., 54:475-478 (1986)].
Circulating immune complexes may block or reduce the efficiency of the natural effector mechanisms of the immune system, as has been postulated for malignant transformations [See Feldman, et al., J. Exp. Med., 131:247 (1970); Mingari, et al., J. Immunol., 121:767 (1978); Theofilopoulos, et al., J. Immunol., 119:657-663 (1977); Theofilopoulos, et al., Immunodiagnostics of Cancer, p. 896, M. Decker, New York (1979), and Levinsky, et al., Lancet, 1:564 (1977)]. The removal of immune complexes from the circulation may reduce many of the clinical problems associated with autoimmune and infectious diseases and cancer. For example, it is beneficial to monitor circulating immune complex levels in blood and to specifically bind and remove circulating immune complexes which may otherwise compromise immune system function or lead to acute or chronic inflammation. Accordingly, many investigators have approached the monitoring and removal of circulating immune complexes by devising assays and adsorbents to selectively react with circulating immune complexes while not reacting with uncomplexed immunoglobulins.
Methods used to assay for immune complexes include: physical separation using polyethylene glycol; reducing the temperature of the solution containing the immune complexes (i.e., cold precipitation); binding of the immune complexes to complement protein C1q or to antibodies specific to the complement protein C3 and C3 degradation products; binding of the immune complexes to rheumatoid factors; binding to the bovine protein conglutinin; or binding of the immune complexes to platelets or to the Raji lymphoblastoid cell line [See Theofilopoulos, et al., Hosp. Pract., 107-121 (February 1980); Ingram, Animal Models of Immunological Processes, pp. 221-253 (1982); Levinson, et al., J. Clin. Immunoassay, 7:328-336 (1984); Singh, et al., J. Immunol. Meth., 50:109-114 (1982); Hay, et al., Clin. Ext. Immunol., 24:396-400 (1976); Pereira, et al., J. Immunol., 125:763-770 (1980); Theofilopoulos, et al., J. Clin. Invest., 61:1570 (1978); Creighton, et al., J. Immunol., 111:1219 (1973); Schur, N. Engl. J. Med., 298:161 (1978), and Bruneau, et al., J. Clin. Invest., 64:191 (1979)]. There are advantages to each assay, as well as disadvantages which include insensitivity, nonspecificity, inability to detect immune complexes of all sizes and of all immunoglobulin isotypes and subisotypes, reliance on immune complexes containing complement proteins, and interference by non-complexed immunoglobulins. It also appears that the ability to detect circulating immune complexes in serum from a patient with a specific disease varies with the assay used. [See McDougal, J. S., et al., Adv. Clin. Chem., 24:1-60 (1985)]. Therefore, results gathered by any of the aforementioned assays may be considered to be of marginal value for diagnostic purposes, but may be used to support a diagnosis, to assess disease severity by correlating with amounts of immune complexes, or to monitor follow-up after therapeutic treatment as suggested by Feldkamp in Clin. Chem. News, 3:5-6 (1987).
Methods of removing circulating immune complexes from blood have emphasized a plasma exchange or blood filtration therapy. Generally, for blood filtration therapy, a substance which can bind complexed immunoglobulin is immobilized on a solid support which is encased online, through which the blood or plasma removed from the patient is passed. In some cases, after passage over the adsorbent (in an extracorporeal device), blood components are reinfused into the patient thus eliminating the need for large amount of replacement plasma or other replacement fluids.
Currently, there is interest in study of the use of Staphlococcal Protein A (the various subtypes being collectively known as "Protein A") as the immobilized circulating immune complex adsorbent. Some trials by investigators have resulted in promising results while others have failed, presumably due to Protein A's inability to effectively differentiate immunoglobulin in immune complexes from noncomplexed immunoglobulin and to whatever effect this bacterial product has on stimulating pyrogenic activity, complement activation and general immune system reactivity as reported by Betram, et al., J. Biol. Resp. Mod., 3:235-240 (1984); Terman, et al., New Engl. J. Med., 305:1195-2000 (1981); Dobre, et al., J. Immunol. Meth, 66:171-178 (1984); Nilsson, et al., Scand. J. Haematol., 30:458-464 (1983), and Nauts, in Host Defense Against Cancer and Its Potentiation, pp. 337-351 (1975).
Protein A binds to the "Fc" portion of immunoglobulins. Other investigators have proposed or attempted using other Fc receptors, such as C1q or rheumatoid factor, or a specific antigen or antibody, as immobilized immunoadsorbents. [See Nilsson, I. M., et al., Plasma. Ther. Transfers. Technol., 5:127-134 (1984); Lai, K. N., et al., Artificial Organs, 11:259-264 (1987); Nilsson, I. M., et al., In Factor VIII Inhibitors, p. 225 (1984); Liberti, P. A., et al., J. Immunol., 123:2212-2219 (1979); Randerson, D. H., et al., Artificial Organs, 6:43-49 (1982)].
C-reactive protein (CRP) is the prototype acute phase reactant. It was first described by Tillett and Francis in J. Exp. Med., 52:561-571 (1930), who observed that sera from acutely ill patients precipitated with fraction C of the pneumococcal cell wall. Others subsequently identified the reactive serum factor as protein, hence the designation C-reactive protein.
It was later discovered that CRP has a role in host defense. CRP can recognize and bind one of several ligands on the cell or bacterial surface, or in suspension. These ligands include phosphorylcholine, chromatin and polycations. It appears that certain CRP-ligand complexes have the capacity to activate the complement pathway, thus stimulating certain aspects of the immune system.
In clinical use, CRP has been used as a marker of acute inflammation. Its exact role in the body's inflammatory response is not yet known, although it has been shown that injecting mice with liposomes containing C-reactive protein can be effective in inhibiting or reversing certain tumor growth [See Deodhar, S. D., et al., Cancer Res., 42:5084-5088 (1982); Barna, B. P., et al., Cancer Res., 44:305-310 (1984); Thombre, P. S., et al., Cancer Immunol. Immunother., 16:145-150 (1984)].
Under particular experimental conditions, CRP can be altered so as to have charge, size, solubility and antigenicity characteristics significantly different than the CRP molecule monitored as a marker of acute inflammation [See Potempa, L. A., et al., Mol. Immunol. 20:1165-1175 (1983)]. The distinctive antigenicity associated with altered CRP has been referred to as "neo-CRP," and the altered CRP molecule itself has been referred to as "modified-CRP." Using appropriate reagents and assays, it has been determined that modified-CRP expressing neo-CRP antigenicity functioned in vitro in a variety of assays used to assess the state of the immune system reactivity. In brief, modified-CRP was found to:
stimulate glass-adherent monocytes to secrete interleukin 1; PA1 stimulate glass-adherent monocytes to increase prostaglandin and leukotriene metabolites; PA1 stimulate glass-adherent monocytes to increase a lymphoblastogenesis response to autologous lymphocytes; PA1 stimulate platelets to aggregate and secrete granular constituents; PA1 potentiate polymorphonuclear leukocytes and monocytes to in crease oxidative metabolism stimulated by aggregated (complexed) immunoglobulins; and PA1 stimulate endothelial cells to proliferate and synthesize proteins.
[See Potempa, L. A., Gewurz, H., Harris, J. E., and Braun, D. P., Protides of the Biological Fluids, Vol. 34, pp. 287-290 (1986); Potempa, L. A., Zeller, J. M., Fiedel, B. A., Kinoshita, C. M. and Gewurz, H., Inflammation, 12:391-405 (1988); Gupta, R. C., Potempa, L. A., Krishnan, M. R., and Postlethwaite, A. E., Arthritis & Rheumatism, 31: R39a (1988); Chu, E. B., Potempa, L. A., Harris, J. E., Gewurz, H., and Braun, D. P., Amer. Assoc. Cancer Res. 29: 371a (1988); Doughery, T. J., Zeller, J. M., Potempa, L. A., Gewurz, H., and Siegal, J., Protides of the Biological Fluids, Vol. 34, pp. 291-293 (1986)].
The exact mechanism by which modified-CRP contributes to these activities is unknown. However, a consistent feature common to all these listed activities is that modified-CRP either mimics or enhances those activities stimulated by aggregated immunoglobulin.
Also, modified-CRP has also been used in vivo. Mice injected with modified-CRP 30 min. prior to receiving a lethal dose (90%) of type 7F Streptococcus pneumoniae survived death in a significant and dose-related manner [See Chudwin, D. S., et al., J. Allergy Clin. Immunol., 77:216a (1986)].
Using appropriate reagents and techniques to identify the natural occurrence of modified-CRP as the neo-CRP antigen on body cells, it has recently been determined that neo-CRP antigenicity is found as a natural component on the surface of large granular lymphocytes (i.e., natural killer (NK) cells, B lymphocytes, polymorphonuclear leukocytes and monocytes). On natural killer and B-cells, the antigen recognized by antibody to neo-CRP has been found to be directly associated with the Fc receptors found on either cell. The Fc receptor on natural killer cells is distinctive from the Fc receptor on B-cells, suggesting that CRP may be a common link in the structures of two physically distinct but functionally related molecules [See Unkeless, J. C., et al., Adv. Immunol., 31:247-270 (1981); Perussia, B., et al., J. Immunol., 133:180-189 (1984); Anderson, C. L. and Looney, J. R., Immunol. Today, 7:264-266 (1986)].